A quantitative assay for assessing allelic proportions by iterative gap ligation.
A variety of techniques are currently available for detecting point mutations in DNA. These techniques are frequently not sensitive enough to be applied as quantitative assays in evaluation of relative occurrence of alleles in cases of polymorphism or when variations in allelic gene expression are being evaluated at the level of RNA. We report here the establishment of an iterative gap ligation (IGL) assay that is both quantitative and sensitive. The design of the assay is such that ligation of an upstream to a downstream primer across a single nucleotide gap will only occur if the gap is filled with a deoxynucleotide complementary to the wild-type or mutant sequence. Under conditions in which excess upstream primer saturates the template concurrently with limiting amounts of downstream primer quantitative ligation is absolutely dependent on provision of the appropriate gap filling nucleotide. When gap ligation occurs in a single incubation, or cycle, the amount of ligated product is a linear function of the relative amount of mutant sequence, with a sensitivity and detection limit of approximately 3% over a range of relative concentrations of 0-100%. When the reaction occurs over multiple cycles, or iterations, gap ligation becomes a non-linear function such that small changes in the relative proportions of alleles produce a disproportionately large amount of ligation. As a consequence, the sensitivity and limits of detection of the assay improve to 0.2% after only 8 cycles. The development of this assay provides a unique means of quantifying allelic polymorphisms in both DNA and RNA (after initial amplification by PCR or RT-PCR) and should be applicable to any experimental settings in which nucleic acids from tissues or mixed populations of cells are being evaluated.
Stewart
J
J
Department of Pathology and Laboratory Medicine, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Kozlowski
P
P
Sowden
M
M
Messing
E
E
Smith
H C
HC
D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D011088:DNA Ligases; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D016162:Genes, Wilms Tumor; D005821:Genetic Techniques / Q000706:statistics & numerical data; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004247:DNA; D011088:DNA Ligases